Detection, identification and differentiation of eubacterial taxa using a hybridization assay
The present invention relates to a method for the specific detection and/or identification of Enterococcus species, in particular Enterococcus faecalis and/or Enterococcus faecium , using new nucleic acid sequences derived from the ITS (Internal Transcribed Spacer) region. The present invention relates also to said new nucleic acid sequences derived from the ITS region, between the 16S and 23S ribosomal ribonucleic acid (rRNA) or rRNA genes, to be used for the specific detection and/or identification of Enterococcus species, in particular of Enterococcus faecalis and/or Enterococcus faecium , in a biological sample. It relates also to nucleic acid primers to be used for the amplification of said spacer region of Enterococcus species in a sample.
1. A set of two or three polynucleotide probes, said set of two or three polynucleotide probes consisting of any combination of the sequences of SEQ ID NOs: 29 and 68; 34 and 69; 35 and 73; 82 and 73; 36, 56 and 73, wherein at least one of said probes is labeled by at least one marker selected from the group consisting of 32 P, 35 S, biotin, digoxogenin, fluorescent dye and an enzyme.
2. A method for detecting and/or identifying Enterococcus species in a sample using at least one set of polynucleotide probes according to claim 1 comprising:
(i) if need be releasing, isolating and/or concentrating the polynucleic acids in the sample;
(ii) if need be amplifying the 16S-23S rRNA spacer region, or a fragment comprising the target sequence, or the target sequence or a fragment thereof, with at least one suitable primer pair;
(iii) hybridizing specifically the polynucleic acids of step (i) or (ii) with said at least one set of polynucleotide probes;
(iv) detecting the hybrids formed; and
(v) interpreting the signal(s) obtained and inferring the presence of Enterococcus species and/or identifying the Enterococcus species in the sample.
3. A method according to claim 2 , wherein said suitable primer pair consists of any combination of a forward primer polynucleotide selected from the group consisting of SEQ ID NO 3, 4, 5, 6, 7, 8, 9, 10, 11 and their homologues, and a reverse primer polynucleotide selected from the group consisting of SEQ ID NO 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and their homologues.
4. A method according to claim 3 , wherein two polynucleotide probes consisting of any combination of the sequences of SEQ ID NOs: 29 and 68; 34 and 69; 35 and 73; 82 and 73 are used.
5. A kit for detection and/or identification of Enterococcus species comprising the following components:
at least one set of two or three polynucleotide probes of claim 1 and
a hybridization buffer or components necessary for producing said buffer.